The growth and maturation of mammalian germ cells is intricately controlled by hormones; including gonadotropins secreted by the anterior pituitary; and local paracrine factors. The majority of the oocytes within the adult human ovary are maintained in prolonged stage of first meiotic prophase; enveloped by surrounding follicular somatic cells. Periodically, a group of primordial follicles enters a stage of follicular growth. During this time, the oocyte undergoes a large increase in volume, and the number of follicular granulosa cells increase. The maturing oocyte synthesizes paracrine factors that allow the follicle cells to proliferate, and the follicle cells secrete growth and differentiation factors (for example TGF-β2, VEGF, leptin, and FGF2) that enhance angiogenesis and allow the oocyte to grow. After progressing to a certain stage, oocytes and their follicles die, unless they are exposed to gonadotropic hormones that prevent somatic cell apoptosis.
Mammalian ovaries consist of follicles as basic functional units. The total number of ovarian follicles is determined early in life, and the depletion of this pool leads to reproductive senescence. Each follicle develops to either ovulate or, more likely, to undergo degeneration. Individual follicles consist of an innermost oocyte, surrounding granulosa cells, and outer layers of thecal cells. The fate of each follicle is controlled by endocrine as well as paracrine factors. The follicles develop through primordial, primary, and secondary stages before acquiring an antral cavity. At the antral stage a few follicles, under the cyclic gonadotropin stimulation that occurs after puberty, reach the preovulatory stage and become a major source of the cyclic secretion of ovarian estrogens in women of reproductive age. In response to preovulatory gonadotropin surges during each reproductive cycle, the dominant Graafian follicle ovulates to release the mature oocyte for fertilization, whereas the remaining theca and granulosa cells undergo transformation to become the corpus luteum.
Once entering the growing pool, ovarian follicles continue to progress into primary, secondary, and early antral stages with minimal loss. Although FSH treatment is widely used to generate preovulatory follicles in infertile patients mainly by suppressing the apoptosis of early antral follicles, some patients are low responders to FSH treatment because their ovaries contain few early antral follicles as reflected by their elevated serum FSH and lower AMH levels on day 3 of the menstrual cycle.
Throughout the reproductive life, primordial follicles undergo initial recruitment to enter the growing pool of primary follicles. In the human ovary, greater than 120 days are required for the primary follicles to reach the secondary follicle stage, whereas 71 days are needed to grow from the secondary to the early antral stage. Once initiated to enter the growing pool, ovarian follicles progress to reach the antral stage and minimal follicle loss was found until the early antral stage. During cyclic recruitment, increases in circulating FSH allow a cohort of antral follicles to escape apoptotic demise. Among this cohort, a leading follicle emerges as dominant by secreting high levels of estrogens and inhibins to suppress pituitary FSH release. The result is a negative selection of the remaining cohort, leading to its ultimate demise. Concomitantly, increases in local growth factors and vasculature allow a positive selection of the dominant follicle, thus ensuring its final growth and eventual ovulation and luteinization. After cyclic recruitment, it takes only 2 weeks for an antral follicle to become a dominant Graafian follicle. The overall development of human follicles from primordial to preovulatory stages require more than six months.
The development of follicles from the smallest primordial and primary follicles to the largest preovulatory follicles requires different stage-dependent stimulatory and survival factors. FSH, activin, nerve growth factor, and GDF-9 are important for the growth and differentiation of primary and/or secondary follicles. The growth of antral and preovulatory follicles is dependent on gonadotropin stimulation, and FSH is a major survival factor to rescue early antral follicles from apoptotic demise during cyclic recruitment. Treatment with FSHctp (a long-acting FSH agonist) has resulted in increased ovarian weight and follicle development. Thus, the development of follicles can be divided into gonadotropin-dependent and gonadotropin-responsive stages.
Methods of efficiently maturing ovarian follicles from primary through secondary, antral, and preovulatory stages is of great interest, including methods for in vitro follicle maturation. The present invention addresses this issue.
Publications
Harwood et al. (2008) Dev Dyn 237:1099-1111, Members of the WNT signaling pathways are widely expressed in mouse ovaries, oocytes, and cleavage stage embryos. Fan et al. (2010) Mol Endocrinol 24:1529-1542, Beta-catenin (CTNNB1) promotes preovulatory follicular development but represses LH-mediated ovulation and luteinization. Kim et al. (2008) Mol Biol Cell 19:2588-2596, R-Spondin family members regulate the Wnt pathway by a common mechanism. McGee et al. (1997) Biol Reprod 57:990-998, Follicle-stimulating hormone enhances the development of preantral follicles in juvenile rats. Tarlatzis et al. (2003) Hum Reprod Update 9:61-76, Clinical management of low ovarian response to stimulation for IVF: a systematic review. Chassot et al. (2008) Hum Mol Genet. 17:1264-1277, Activation of beta-catenin signaling by Rspo1 controls differentiation of the mammalian ovary. Tomizuka et al. (2008) Hum Mol Genet. 17:1278-1291, R-spondin1 plays an essential role in ovarian development through positively regulating Wnt-4 signaling. Parma et al. (2006) Nat Genet. 38:1304-1309 R-spondin1 is essential in sex determination, skin differentiation and malignancy.
Sudoh et al. (1990) Biochem Biophys Res Commun 168:863-870, C-type natriuretic peptide (CNP): a new member of natriuretic peptide family identified in porcine brain. Koller et al. (1991) Science 252:120-123, Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Jankowski et al. (1997) Biol Reprod 56:59-66, C-type natriuretic peptide and the guanylyl cyclase receptors in the rat ovary are modulated by the estrous cycle. Gutkowska et al. (1999) Biol Reprod 61:162-170, Hormonal regulation of natriuretic peptide system during induced ovarian follicular development in the rat. Zhang et al. (2010), Science 330:366-369 Granulosa cell ligand NPPC and its receptor NPR2 maintain meiotic arrest in mouse oocytes. Li et al. (2010) Proc Natl Acad Sci USA 107:10280-10284, Activation of dormant ovarian follicles to generate mature eggs. Kawamura et al. (2005) Proc Natl Acad Sci USA 102:9206-9211, Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos.